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bioRxiv · 10.1101/2024.09.29.615683

Radical Protein Footprinting in Mammalian Whole Blood

Tobin, L.·Misra, S. K.·Liu, H.·Jones, L. M.·Sharp, J. S.
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Abstract

Hydroxyl Radical Protein Footprinting (HRPF) is a powerful tool to probe protein higher-order structure, as well as protein-protein and protein-carbohydrate interactions. It is mostly performed in vitro, but recent advances have extended its use to live cells, nematodes, and 3D cultures. However, application in living mammalian tissues has not been accomplished. Here, we present the first successful use of radical protein footprinting (RPF) in mammalian whole blood from wild-type (WT) and type 2 diabetes mellitus (T2DM) BKS. Cg Dock7m +/+ Leprdb/J mice. Using persulfate photoactivated with the FOX Photolysis System, we achieved effective protein labeling without significant disruption to blood cell morphology. An optimized quenching protocol eliminated background labeling. We report oxidative modifications in 11 selected proteins, revealing disease-associated conformational changes in multiple proteins. These findings demonstrate the feasibility of RPF in mammalian blood and open new opportunities for structural proteomics in preclinical models and clinical samples.

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Tobin, L., Misra, S. K., Liu, H., Jones, L. M., Sharp, J. S.. 2024-09-29. Radical Protein Footprinting in Mammalian Whole Blood. https://doi.org/10.1101/2024.09.29.615683

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